Processing polynucleotide-containing samples

ABSTRACT

Methods and systems for processing polynucleotides (e.g., DNA) are disclosed. A processing region includes one or more surfaces (e.g., particle surfaces) modified with ligands that regain polynucleotides under a first set of conditions (e.g., temperature and pH) and release the polynucleotides under a second set of conditions (e.g., higher temperature and/or more basic pH). The processing region can be used to, for example, concentrate polynucleotides of a sample and/or separate inhibitors of amplification reactions from the polynucleotides. Microfluidic devices with a processing region are disclosed.

RELATED APPLICATIONS

This application claims the benefit of U.S. provisional application No.60/567,174, filed May 3, 2004 and U.S. provisional application No.60/645,784, filed Jan. 21, 2005, both of which applications areincorporated herein by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates to methods for processingpolynucleotide-containing samples as well as to related systems.

BACKGROUND

The analysis of a biological sample often includes detecting one or morepolynucleotides present in the sample. One example of detection isqualitative detection, which relates, e.g., to the determination of thepresence of the polynucleotide and/or the determination of informationrelated to, e.g., the type, size, presence or absence of mutations,and/or the sequence of the polynucleotide. Another example of detectionis quantitative detection, which relates, e.g., to the determination ofthe amount of polynucleotide present. Detection may include bothqualitative and quantitative aspects.

Detecting polynucleotides often involves the use of an enzyme. Forexample, some detection methods include polynucleotide amplification bypolymerase chain reaction (PCR) or a related amplification technique.Other detection methods that do not amplify the polynucleotide to bedetected also make use of enzymes. However, the functioning of enzymesused in such techniques may be inhibited by the presence of inhibitorspresent along with the polynucleotide to be detected. The inhibitors mayinterfere with, for example, the efficiency and/or specificity of theenzymes.

SUMMARY OF THE INVENTION

One aspect of the present invention relates to a method and relatedsystems for processing one or more polynucleotide(s) (e.g., toconcentrate the polynucleotide(s) and/or to separate the polynucleotidesfrom inhibitor compounds (e.g., hemoglobin) that might inhibit detectionand/or amplification of the polynucleotides).

In some embodiments, the method includes contacting the polynucleotidesand a relatively immobilized compound that preferentially associateswith (e.g., retains) the polynucleotides as opposed to inhibitors. Anexemplary compound is a poly-cationic polyamide (e.g., poly-L-lysineand/or the poly-D-lysine), which may be bound to a surface (e.g., asurface of one or more particles). The compound retains thepolynucleotides so that the polynucleotides and inhibitors may beseparated, such as by washing the surface with the compound andassociated polynucleotides. Upon separation, the association between thepolynucleotide and compound may be disrupted to release (e.g., separate)the polynucleotides from the compound and surface.

In some embodiments, the surface (e.g., a surface of one or moreparticles) is modified with a poly-cationic polyamide, which may becovalently bound to the surface. The polycationic polyamide may includeat least one of poly-L-lysine and poly-D-lysine. In some embodiments,the poly-cationic polyamide (e.g., the at least one of the poly-L-lysineand the poly-D-lysine) have an average molecular weight of at leastabout 7500 Da. The poly-cationic polyamide (e.g., the at least one ofthe poly-L-lysine and the poly-D-lysine) may have an average molecularweight of less than about 35,000 Da (e.g., an average molecular weightof less than about 30000 Da (e.g., an average molecular weight of about25,000 Da)). The poly-cationic polyamide (e.g., the at least one of thepoly-L-lysine and the poly-D-lysine) may have a median molecular weightof at least about 15,000 Da. The poly-cationic polyamide (e.g., the atleast one of the poly-L-lysine and the poly-D-lysine) may have a medianmolecular weight of less than about 25,000 Da (e.g., a median molecularweight of less than about 20,000 Da (e.g., a median molecular weight ofabout 20,000 Da).

Another aspect of the invention relates to a sample preparation deviceincluding a surface including a poly-cationic polyamide bound theretoand a sample introduction passage in communication with the surface forcontacting the surface with a fluidic sample.

In some embodiments, the device includes a heat source configured toheat an aqueous liquid in contact with the surface to at least about 65°C.

In some embodiments, the device includes a reservoir of liquid having apH of at least about 10 (e.g., about 10.5 or more). The device isconfigured to contact the surface with the liquid (e.g., by actuating apressure source to move the liquid).

In some embodiments, the surface comprises surfaces of a plurality ofparticles.

In some embodiments, the poly-cationic polyamide includes poly-L-lysineand/or poly-D-lysine.

Another aspect of the invention relates to a method for processing asample including providing a mixture including a liquid and an amount ofpolynucleotide, contacting a retention member with the mixture. Theretention member may be configured to preferentially retainpolynucleotides as compared to polymerase chain reaction inhibitors.Substantially all of the liquid in the mixture is removed from theretention member. The polynucleotides are released from the retentionmember.

The polynucleotide may have a size of less than about 7.5 Mbp.

The liquid may be a first liquid and removing substantially all of theliquid from the retention member may include contacting the retentionmember with a second liquid.

Contacting the retention member with a second liquid can includeactuating a thermally actuated pressure source to apply a pressure tothe second liquid. Contacting the retention member with a second liquidcan include opening a thermally actuated valve to place the secondliquid in fluid communication with the retention member.

The second liquid may have a volume of less than about 50 microliters.

The retention member may include a surface having a compound configuredto bind polynucleotides preferentially to polymerase chain reactioninhibitors (e.g., hemoglobin, peptides, faecal compounds, humic acids,mucousol compounds, DNA binding proteins, or a saccharide).

The surface may include a poly-lysine (e.g., poly-L-lysine and/orpoly-D-lysine).

The second liquid may include a detergent (e.g., SDS).

Releasing may include heating the retention member to a temperature ofat least about 50° C. (e.g., at about 65° C.). The temperature may beinsufficient to boil the liquid in the presence of the retention memberduring heating. The temperature may be 100° C. or less (e.g., less than100° C., about 97° C. or less). The temperature may be maintained forless than about 10 minutes (e.g., for less than about 5 minutes, forless than about 3 minutes).

The releasing may be performed without centrifugation of the retentionmember.

In certain embodiments, PCR inhibitors are rapidly removed from clinicalsamples to create a PCR-ready sample. The method may comprise thepreparation of a polynucleotide-containing sample that is substantiallyfree of inhibitors. The samples may be prepared from, e.g., crudelysates resulting from thermal, chemical, ultrasonic, mechanical,electrostatic, and other lysing techniques. The samples may be preparedwithout centrifugation. The samples may be prepared using microfluidicdevices or on a larger scale.

Another aspect of the invention relates to a retention member, e.g., aplurality of particles such as beads, comprising bound poly-lysine,e.g., poly-L-lysine, and related methods and systems. The retentionmember preferentially binds polynucleotides, e.g., DNA, as compared toinhibitors. The retention member may be used to prepare polynucleotidessamples for further processing, such as amplification by polymerasechain reaction.

In certain embodiments, more than 90% of a polynucleotide present in asample may be bound to the retention member, released, and recovered.

In certain embodiments, a polynucleotide may be bound to the retentionmember, released, and recovered, in less than 10 minutes, less than 7.5minutes, less than 5 minutes, or less than 3 minutes.

A polynucleotide may be bound to a retention member, released, andrecovered without subjecting the polynucleotide, retention member,and/or inhibitors to centrifugation.

Separating the polynucleotides and inhibitors generally excludessubjecting the polynucleotides, inhibitors, processing region, and/orretention member to sedimentation (e.g., centrifugation).

Another aspect of the invention relates to a microfluidic deviceincluding a channel, a first mass of a thermally responsive substance(TRS) disposed on a first side of the channel, a second mass of a TRSdisposed on a second side of the channel opposite the first side of thechannel, a gas pressure source associated with the first mass of theTRS. Actuation of the gas pressure source drives the first mass of theTRS into the second mass of the TRS and obstructs the channel.

The microfluidic device can include a second gas pressure sourceassociated with the second mass of the TRS. Actuation of the second gaspressure source drives the second mass of TRS into the first mass ofTRS.

At least one (e.g., both) of the first and second masses of TRS may be awax.

Another aspect of the invention relates to a method for obstructing achannel of a microfluidic device. A mass of a TRS is heated and drivenacross the channel (e.g., by gas pressure) into a second mass of TRS.The second mass of TRS may also be driven (e.g., by gas pressure) towardthe first mass of TRS.

Another aspect of the invention relates to an actuator for amicrofluidic device. The actuator includes a channel, a chamberconnected to the channel, at least one reservoir of encapsulated liquiddisposed in the chamber, and a gas surrounding the reservoir within thechamber. Heating the chamber expands the reservoir of encapsulatedliquid and pressurizes the gas. Typically the liquid has a boiling pointof about 90° C. or less. The liquid may be a hydrocarbon having about 10carbon atoms or fewer.

The liquid may be encapsulated by a polymer.

The actuator may include multiple reservoirs of encapsulated liquiddisposed in the chamber.

The multiple reservoirs may be dispersed within a solid (e.g., a wax).

The multiple reservoirs may be disposed within a flexible enclosure(e.g., a flexible sack).

Another aspect of the invention relates to a method includingpressurizing a gas within a chamber of a microfluidic to create a gaspressure sufficient to move a liquid within a channel of themicrofluidic device. Pressurizing the gas typically expanding at leastone reservoir of encapsulated liquid disposed within the chamber.

Expanding the at least one reservoir can include heating the chamber.

Pressurizing the gas can include expanding multiple reservoirs ofencapsulated liquid.

Another aspect of the invention relates to a method for combining (e.g.,mixing) first and second liquids and related devices. The deviceincludes a mass of a temperature responsive substance (TRS) thatseparates first and second channels of the device. The device isconfigured to move a first liquid along the first channel so that aportion (e.g., a medial portion) of the first liquid is adjacent the TRSand to move a second liquid along the second channel so that a portion(e.g., a medial portion) of second liquid is adjacent the TRS. A heatsource is actuated to move the TRS (e.g., by melting, dispersing,fragmenting). The medial portions of the first and second liquidstypically combine without being separated by a gas interface. Typically,only a subset of the first liquid and a subset of the second liquid arecombined. The liquids mix upon being moved along a mixing channel.

Another aspect of the invention relates to a lyophilized reagentparticle and a method of making the particle.

In some embodiments, the lyophilized particles include multiple smallerparticles each having a plurality of ligands that preferentiallyassociate with polynucleotides as compared to PCR inhibitors. Thelyophilized particles can also (or alternatively) include lysingreagents (e.g., enzymes) configured to lyse cells to releasepolynucleotides. The lyophilized particles can also (or alternatively)include enzymes (e.g., proteases) that degrade proteins.

Cells can be lysed by combining a solution of the cells with thelyophilized particles to reconstitute the particles. The reconstitutedlysing reagents lyse the cells. The polynucleotides associate withligands of the smaller particles. During lysis, the solution may beheated (e.g., radiatively using a lamp (e.g., a heat lamp).

In some embodiments, lyophilized particles include reagents (e.g.,primers, control plasmids, polymerase enzymes) for performing a PCRreaction.

A method for making lyophilized particles includes forming a solution ofreagents of the particle and a cryoprotectant (e.g., a sugar orpoly-alcohol). The solution is deposited dropwise on a chilledhydrophobic surface (e.g., a diamond film or polytetrafluoroethylenesurface). The particles freeze and are subjected to reduced pressure(typically while still frozen) for a time sufficient to remove (e.g.,sublimate) the solvent. The lyophilized particles may have a diameter ofabout 5 mm or less (e.g., about 2.5 mm or less, about 1.75 mm or less).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of a microfluidic device.

FIG. 2 is a cross-sectional view of a processing region for retainingpolynucleotides and/or separating polynucleotides from inhibitors.

FIG. 3. is a cross-sectional view of an actuator.

FIG. 4 is a perspective view of a microfluidic device.

FIG. 5 is a side cross-sectional view of the microfluidic device of FIG.4.

FIG. 6 is a perspective view of a microfluidic network of themicrofluidic device of FIG. 4.

FIG. 7 illustrates an array of heat sources for operating components ofthe microfluidic device of FIG. 4.

FIGS. 8 and 9 illustrate a valve in the open and closed statesrespectively.

FIGS. 10A-10D illustrate a mixing gate of the microfluidic network ofFIG. 6 and adjacent regions of the network.

FIG. 11 illustrates a device for separating polynucleotides andinhibitors.

FIG. 12 illustrates the device of FIG. 11 and a device for operationthereof.

FIG. 13 illustrates a microfluidic device.

FIG. 14 is a cross-section of the microfluidic device of FIG. 13 takenalong 5.

FIG. 15 illustrates the retention of herring sperm DNA.

FIG. 16 illustrates the retention and release of DNA from group Bstreptococci;

FIG. 17 illustrates the PCR response of a sample from which inhibitorshad been removed and of a sample from which inhibitors had not beenremoved.

FIG. 18 illustrates the PCR response of a sample prepared in accord withthe invention and a sample prepared using a commercial DNA extractionmethod.

FIG. 19 a illustrates a flow chart showing steps performed during amethod for separation polynucleotides and inhibitors.

FIG. 19 b illustrates DNA from samples subjected to the method of FIG.19 a.

DETAILED DESCRIPTION OF THE INVENTION

Analysis of biological samples often includes determining whether one ormore polynucleotides (e.g., a DNA, RNA, mRNA, or rRNA) is present in thesample. For example, one may analyze a sample to determine whether apolynucleotide indicative of the presence of a particular pathogen ispresent. Typically, biological samples are complex mixtures. Forexample, a sample may be provided as a blood sample, a tissue sample(e.g., a swab of, for example, nasal, buccal, anal, or vaginal tissue),a biopsy aspirate, a lysate, as fungi, or as bacteria. Polynucleotidesto be determined may be contained within particles (e.g., cells (e.g.,white blood cells and/or red blood cells), tissue fragments, bacteria(e.g., gram positive bacteria and/or gram negative bacteria), fungi,spores). One or more liquids (e.g., water, a buffer, blood, bloodplasma, saliva, urine, spinal fluid, or organic solvent) is typicallypart of the sample and/or is added to the sample during a processingstep.

Methods for analyzing biological samples include providing a biologicalsample (e.g., a swab), releasing polynucleotides from particles (e.g.,bacteria) of the sample, amplifying one or more of the releasedpolynucleotides (e.g., by polymerase chain reaction (PCR)), anddetermining the presence (or absence) of the amplified polynucleotide(s)(e.g., by fluorescence detection). Biological samples, however,typically include inhibitors (e.g., mucousal compounds, hemoglobin,faecal compounds, and DNA binding proteins) that can inhibit determiningthe presence of polynucleotides in the sample. For example, suchinhibitors can reduce the amplification efficiency of polynucleotides byPCR and other enzymatic techniques for determining the presence ofpolynucleotides. If the concentration of inhibitors is not reducedrelative to the polynucleotides to be determined, the analysis canproduce false negative results.

We describe methods and related systems for processing biologicalsamples (e.g., samples having one or more polynucleotides to bedetermined). Typically, the methods and systems reduce the concentrationof inhibitors relative to the concentration of polynucleotides to bedetermined.

Referring to FIG. 1, a microfluidic device 200 includes first, second,and third layers 205, 207, and 209 that define a microfluidic network201 having various components configured to process a sample includingone or more polynucleotides to be determined. Device 200 typicallyprocesses the sample by increasing the concentration of a polynucleotideto be determined and/or by reducing the concentration of inhibitorsrelative to the concentration of polynucleotide to be determined.

We now discuss the arrangement of components of network 201.

Network 201 includes an inlet 202 by which sample material can beintroduced to the network and an output 236 by which a processed samplecan be removed (e.g., expelled by or extracted from) network 201. Achannel 204 extends between inlet 202 and a junction 255. A valve 205 ispositioned along channel 204. A reservoir channel 240 extends betweenjunction 255 and an actuator 244. Gates 242 and 246 are positioned alongchannel 240. A channel 257 extends between junction 255 and a junction257. A valve 208 is positioned along channel 257. A reservoir channel246 extends between junction 259 and an actuator 248. Gates 250 and 252are positioned along channel 246. A channel 261 extends between junction259 and a junction 263. A valve 210 and a hydrophobic vent 212 arepositioned along channel 261. A channel 256 extends between junction 263and an actuator 254. A gate 258 is positioned along channel 256.

A channel 214 extends between junction 263 and a processing chamber 220,which has an inlet 265 and an outlet 267. A channel 228 extends betweenprocessing chamber outlet 267 and a waste reservoir 232. A valve 234 ispositioned along channel 228. A channel 230 extends between processingchamber outlet 267 and output 236.

We turn now to particular components of network 201.

Referring also to FIG. 2, processing chamber 220 includes a plurality ofparticles (e.g., beads, microspheres) 218 configured to retainpolynucleotides of the sample under a first set of conditions (e.g., afirst temperature and/or first pH) and to release the polynucleotidesunder a second set of conditions (e.g., a second, higher temperatureand/or a second, more basic pH). Typically, the polynucleotides areretained preferentially as compared to inhibitors that may be present inthe sample. Particles 218 are configured as a retention member 216(e.g., a column) through which sample material (e.g., polynucleotides)must pass when moving between the inlet 265 and outlet 267 of processingregion 220.

A filter 219 prevents particles 218 from passing downstream ofprocessing region 220. A channel 287 connects filter 219 with outlet267. Filter 219 has a surface area within processing region 220 that islarger than the cross-sectional area of inlet 265. For example, in someembodiments, the ratio of the surface area of filter 219 withinprocessing region 220 to the cross-sectional area of inlet 265 (whichcross sectional area is typically about the same as the cross-sectionalarea of channel 214) is at least about 5 (e.g., at least about 10, atleast about 20, at least about 20). In some embodiments, the surfacearea of filter 219 within processing region 220 is at least about 1 mm²(e.g., at least about 2 mm², at least about 3 mm²). In some embodiments,the cross-sectional area of inlet 265 and/or channel 214 is about 0.25mm² or less (e.g., about 0.2 mm or less, about 0.15 mm² or less, about0.1 mm² or less). The larger surface area presented by filter 219 tomaterial flowing through processing region 220 helps prevent clogging ofthe processing region while avoiding significant increases in the voidvolume (discussed below) of the processing region.

Particles 218 are modified with at least one ligand that retainspolynucleotides (e.g., preferentially as compared to inhibitors).Typically, the ligands retain polynucleotides from liquids having a pHabout 9.5 or less (e.g., about 9.0 or less, about 8.75 or less, about8.5 or less). As a sample solution moves through processing region 220,polynucleotides are retained while the liquid and other solutioncomponents (e.g., inhibitors) are less retained (e.g., not retained) andexit the processing region. In general, the ligands to releasepolynucleotides when the pH is about 10 or greater (e.g., about 10.5 orgreater, about 11.0 or greater). Consequently, polynucleotides can bereleased from the ligand modified particles into the surrounding liquid.

Exemplary ligands include, for example, polyamides (e.g., poly-cationicpolyamides such as poly-L-lysine, poly-D-lysine, poly-DL-ornithine).Other ligands include, for example, intercalators, poly-intercalators,minor groove binders polyamines (e.g., spermidine), homopolymers andcopolymers comprising a plurality of amino acids, and combinationsthereof. In some embodiments, the ligands have an average molecularweight of at least about 5000 Da (e.g., at least about 7500 Da, of atleast about 15000 Da). In some embodiments, the ligands have an averagemolecular weight of about 50000 Da or less (e.g., about 35000, or less,about 27500 Da or less). In some embodiments, the ligand is apoly-lysine ligand attached to the particle surface by an amide bond.

In certain embodiments, the ligands are resistant to enzymaticdegradation, such as degradation by protease enzymes (e.g., mixtures ofendo- and exo-proteases such as pronase) that cleave peptide bonds.Exemplary protease resistant ligands include, for example, poly-D-lysineand other ligands that are enantiomers of ligands susceptible toenzymatic attack.

Particles 218 are typically formed of a material to which the ligandscan be associated. Exemplary materials from which particles 218 can beformed include polymeric materials that can be modified to attach aligand. Typical polymeric materials provide or can be modified toprovide carboxylic groups and/or amino groups available to attachligands. Exemplary polymeric materials include, for example,polystyrene, latex polymers (e.g., polycarboxylate coated latex),polyacrylamide, polyethylene oxide, and derivatives thereof. Polymericmaterials that can used to form particles 218 are described in U.S. Pat.No. 6,235,313 to Mathiowitz et al., which patent is incorporated hereinby reference Other materials include glass, silica, agarose, andamino-propyl-tri-ethoxy-silane (APES) modified materials.

Exemplary particles that can be modified with suitable ligands includecarboxylate particles (e.g., carboxylate modified magnetic beads(Sera-Mag Magnetic Carboxylate modified beads, Part #3008050250,Seradyn) and Polybead carboxylate modified microspheres available fromPolyscience, catalog no. 09850). In some embodiments, the ligandsinclude poly-D-lysine and the beads comprise a polymer (e.g.,polycarboxylate coated latex).

In general, the ratio of mass of particles to the mass ofpolynucleotides retained by the particles is no more than about 25 ormore (e.g., no more than about 20, no more than about 10). For example,in some embodiments, about 1 gram of particles retains about 100milligrams of polynucleotides.

Typically, the total volume of processing region 220 (includingparticles 218) between inlet 265 and filter 219 is about 15 microlitersor less (e.g., about 10 microliters or less, about 5 microliters orless, about 2.5 microliters or less, about 2 microliters or less). In anexemplary embodiment, the total volume of processing region 220 is about2.3 microliters. In some embodiments, particles 218 occupy at leastabout 10 percent (e.g., at least about 15 percent) of the total volumeof processing region 220. In some embodiments, particles 218 occupyabout 75 percent or less (e.g., about 50 percent or less, about 35percent or less) of the total volume of processing chamber 220.

In some embodiments, the volume of processing region 220 that is free tobe occupied by liquid (e.g., the void volume of processing region 220including interstices between particles 218) is about equal to the totalvolume minus the volume occupied by the particles. Typically, the voidvolume of processing region 220 is about 10 microliters or less (e.g.,about 7.5 microliters or less, about 5 microliters or less, about 2.5microliters or less, about 2 microliters or less). In some embodiments,the void volume is about 50 nanoliters or more (e.g., about 100nanoliters or more, about 250 nanoliters or more). In an exemplaryembodiment, the total volume of processing region 220 is about 2.3microliters. For example, in an exemplary embodiment, the total volumeof the processing region is about 2.3 microliters, the volume occupiedby particles is about 0.3 microliters, and the volume free to beoccupied by liquid (void volume) is about 2 microliters.

Particles 218 typically have an average diameter of about 20 microns orless (e.g., about 15 microns or less, about 10 microns or less). In someembodiments, particles 218 have an average diameter of at least about 4microns (e.g., at least about 6 microns, at least about 8 microns).

In some embodiments, a volume of channel 287 between filter 219 andoutlet 267 is substantially smaller than the void volume of processingregion 220. For example, in some embodiments, the volume of channel 287between filter 219 and outlet 267 is about 35% or less (e.g., about 25%or less, about 20% or less) of the void volume. In an exemplaryembodiment, the volume of channel 287 between filter 219 and outlet 267is about 500 microliters.

The particle density is typically at least about 10⁸ particles permilliliter (e.g., about 10⁹ particles per milliliter). For example, aprocessing region with a total volume of about 1 microliter may includeabout 10³ beads.

Filter 219 typically has pores with a width smaller than the diameter ofparticles 218. In an exemplary embodiment, filter 219 has pores havingan average width of about 8 microns and particles 218 have an averagediameter of about 10 microns.

In some embodiments, at least some (e.g., all) of the particles aremagnetic. In alternative embodiments, few (e.g., none) of the particlesare magnetic.

In some embodiments, at least some (e.g., all) the particles are solid.In some embodiments, at least some (e.g., all) the particles are porous(e.g., the particles may have channels extending at least partiallywithin them).

Channels of microfluidic network 201 typically have at least onesub-millimeter cross-sectional dimension. For example, channels ofnetwork 201 may have a width and/or a depth of about 1 mm or less (e.g.,about 750 microns or less, about 500 microns, or less, about 250 micronsor less).

A valve is a component that has a normally open state allowing materialto pass along a channel from a position on one side of the valve (e.g.,upstream of the valve) to a position on the other side of the valve(e.g., downstream of the valve). Upon actuation, the valve transitionsto a closed state that prevents material from passing along the channelfrom one side of the valve to the other. For example, valve 205 includesa mass 251 of a thermally responsive substance (TRS) that is relativelyimmobile at a first temperature and more mobile at a second temperature.A chamber 253 is in gaseous communication with mass 251. Upon heatinggas (e.g., air) in chamber 253 and heating mass 251 of TRS to the secondtemperature, gas pressure within chamber 253 moves mass 251 into channel204 obstructing material from passing therealong. Other valves ofnetwork 201 have the same structure and operate in the same fashion asvalve 205.

A mass of TRS can be an essentially solid mass or an agglomeration ofsmaller particles that cooperate to obstruct the passage. Examples ofTRS's include a eutectic alloy (e.g., a solder), wax (e.g., an olefin),polymers, plastics, and combinations thereof. The first and secondtemperatures are insufficiently high to damage materials, such aspolymer layers of device 200. Generally, the second temperature is lessthan about 90° C. and the first temperature is less than the secondtemperature (e.g., about 70° C. or less).

A gate is a component that has a normally closed state that does notallow material to pass along a channel from a position on one side ofthe gate to another side of the gate. Upon actuation, the gatetransitions to a closed state in which material is permitted to passfrom one side of the gate (e.g., upstream of the gate) to the other sideof the gate (e.g., downstream of the gate). For example, gate 242includes a mass 271 of TRS positioned to obstruct passage of materialbetween junction 255 and channel 240. Upon heating mass 271 to thesecond temperature, the mass changes state (e.g., by melting, bydispersing, by fragmenting, and/or dissolving) to permit passage ofmaterial between junction 255 and channel 240.

The portion of channel 240 between gates 242 and 246 forms a fluidreservoir 279 configured to hold a liquid (e.g., water, an organicliquid, or combination thereof). During storage, gates 242 and 246 limit(e.g., prevent) evaporation of liquid within the fluid reservoir. Duringoperation of device 200, the liquid of reservoir 279 is typically usedas a wash liquid to remove inhibitors from processing region 220 whileleaving polynucleotides associated with particles 218. Typically, thewash liquid is a solution having one or more additional components(e.g., a buffer, chelator, surfactant, a detergent, a base, an acid, ora combination thereof). Exemplary solutions include, for example, asolution of 10-50 mM Tris at pH 8.0, 0.5-2 mM EDTA, and 0.5%-2% SDS, asolution of 10-50 mM Tris at pH 8.0, 0.5 to 2 mM EDTA, and 0.5%-2%Triton X-100.

The portion of channel 246 between gates 250 and 252 form a fluidreservoir 281 configured like reservoir 279 to hold a liquid (e.g., asolution) with limited or no evaporation. During operation of device200, the liquid of reservoir 281 is typically used as a release liquidinto which polynucleotides that had been retained by particles 218 arereleased. An exemplary release liquid is an hydroxide solution (e.g., aNaOH solution) having a concentration of, for example, between about 2mM hydroxide (e.g., about 2 mM NaOH) and about 500 mM hydroxide (e.g.,about 500 mM NaOH). In some embodiments, liquid in reservoir 281 is anhydroxide solution having a concentration of about 25 mM or less (e.g.,an hydroxide concentration of about 15 mM).

Reservoirs 279, 281 typically hold at least about 0.375 microliters ofliquid (e.g., at least about 0.750 microliters, at least about 1.25microliters, at least about 2.5 microliters). In some embodiments,reservoirs 279, 281 hold about 7.5 microliters or less of liquid (e.g.,about 5 microliters or less, about 4 microliters or less, about 3microliters or less).

An actuator is a component that provides a gas pressure that can movematerial (e.g., sample material and/or reagent material) between onelocation of network 201 and another location. For example, referring toFIG. 3, actuator 244 includes a chamber 272 having a mass 273 ofthermally expansive material (TEM) therein. When heated, the TEM expandsdecreasing the free volume within chamber 272 and pressurizing the gas(e.g., air) surrounding mass 273 within chamber 272. Typically, gates246 and 242 are actuated with actuator 244. Consequently, thepressurized gas drives liquid in fluid reservoir 279 towards junction255. In some embodiments, actuator 244 can generate a pressuredifferential of more than about 3 psi (e.g., at least about 4 psi, atleast about 5 psi) between the actuator and junction 255.

The TEM includes a plurality of sealed liquid reservoirs (e.g., spheres)275 dispersed within a carrier 277. Typically, the liquid is a highvapor pressure liquid (e.g., isobutane and/or isopentane) sealed withina casing (e.g., a polymeric casing formed of monomers such as vinylidenechloride, acrylonitrile and methylmethacrylate). Carrier 277 hasproperties (e.g., flexibility and/or an ability to soften (e.g., melt)at higher temperatures) that permit expansion of the reservoirs 275without allowing the reservoirs to pass along channel 240. In someembodiments, carrier 277 is a wax (e.g., an olefin) or a polymer with asuitable glass transition temperature. Typically, the reservoirs make upat least about 25 weight percent (e.g., at least about 35 weightpercent, at least about 50 weight percent) of the TEM. In someembodiments, the reservoirs make up about 75 weight percent or less(e.g., about 65 weight percent or less, about 50 weight percent or less)of the TEM. Suitable sealed liquid reservoirs can be obtained fromExpancel (Akzo Nobel).

When the TEM is heated (e.g., to a temperature of at least about 50° C.(e.g., to at least about 75° C., at least about 90° C.)), the liquidvaporizes and increases the volume of each sealed reservoir and of mass273. Carrier 277 softens allowing mass 273 to expand. Typically, the TEMis heated to a temperature of less than about 150° C. (e.g., about 125°C. or less, about 110° C. or less, about 100° C. or less) duringactuation. In some embodiments, the volume of the TEM expands by atleast about 5 times (e.g., at least about 10 times, at least about 20times, at least about 30 times).

A hydrophobic vent (e.g., vent 212) is a structure that permits gas toexit a channel while limiting (e.g., preventing) liquid from exiting thechannel. Typically, hydrophobic vents include a layer of poroushydrophobic material (e.g., a porous filter such as a porous hydrophobicmembrane from Osmonics) that defines a wall of the channel. As discussedbelow, hydrophobic vents can be used to position a microdroplet ofsample at a desired location within network 201.

Hydrophobic vents typically have a length of at least about 2.5 mm(e.g., at least about 5 mm, at least about 7.5 mm) along a channel. Thelength of the hydrophobic vent is typically at least about 5 times(e.g., at least about 10 times, at least about 20 times) larger than adepth of the channel within the hydrophobic vent. For example, in someembodiments, the channel depth within the hydrophobic vent is about 300microns or less (e.g., about 250 microns or less, about 200 microns orless, about 150 microns or less).

The depth of the channel within the hydrophobic vent is typically about75% or less (e.g., about 65% or less, about 60% or less) of than thedepth of the channel upstream and downstream of the hydrophobic vent.For example, in some embodiments the channel depth within thehydrophobic vent is about 150 microns and the channel depth upstream anddownstream of the hydrophobic vent is about 250 microns.

A width of the channel within the hydrophobic vent is typically at leastabout 25% wider (e.g., at least about 50% wider) than a width of thechannel upstream from the vent and downstream from the vent. Forexample, in an exemplary embodiment, the width of the channel within thehydrophobic vent is about 400 microns and the width of the channelupstream and downstream from the vent is about 250 microns.

Microfluidic device 200 can be fabricated as desired. Typically, layers205, 207, and 209 are formed of a polymeric material. Components ofnetwork 201 are typically formed by molding (e.g., by injection molding)layers 207, 209. Layer 205 is typically a flexible polymeric material(e.g., a laminate) that is secured (e.g., adhesively and/or thermally)to layer 207 to seal components of network 201. Layers 207 and 209 maybe secured to one another using adhesive.

In use, device 200 is typically thermally associated with an array ofheat sources configured to operate the components (e.g., valves, gates,actuators, and processing region 220) of the device. In someembodiments, the heat sources are integral with an operating system,which operates the device during use. The operating system includes aprocessor (e.g., a computer) configured to actuate the heat sourcesaccording to a desired protocol. Processors configured to operatemicrofluidic devices are described in U.S. application Ser. No.09/819,105, filed Mar. 28, 2001, which application is incorporatedherein by reference. In other embodiments, the heat sources are integralwith the device itself.

Device 200 may be operated as follows. Valves of network 201 areconfigured in the open state. Gates of network 201 are configured in theclosed state. A fluidic sample comprising polynucleotides is introducedto network 201 via inlet 202. For example, sample can be introduced witha syringe having a Luer fitting. The syringe provides pressure toinitially move the sample within network 201. Sample passes alongchannels 204, 257, 261, and 214 to inlet 265 of processing region 220.The sample passes through processing region 220, exits via outlet 267,and passes along channel 228 to waste chamber 232. When the trailingedge (e.g., the upstream liquid-gas interface) of the sample reacheshydrophobic vent 212, pressure provided by the introduction device(e.g., the syringe) is released from network 201 stopping further motionof the sample.

Typically, the amount of sample introduced is about 500 microliters orless (e.g., about 250 microliters or less, about 100 microliters orless, about 50 microliters or less, about 25 microliters or less, about10 microliters or less). In some embodiments, the amount of sample isabout 2 microliters or less (e.g., of about 0.5 microliters or less).

Polynucleotides entering processing region 220 pass through intersticesbetween the particles 218. Polynucleotides of the sample contactretention member 216 and are preferentially retained as compared toliquid of the sample and certain other sample components (e.g.,inhibitors). Typically, retention member 220 retains at least about 50%of polynucleotides (at least about 75%, at least about 85%, at leastabout 90%) of the polynucleotides present in the sample that enteredprocessing region 220. Liquid of the sample and inhibitors present inthe sample exit the processing region 220 via outlet 267 and enter wastechamber 232. Processing region is typically at a temperature of about50° C. or less (e.g., 30° C. or less) during introduction of the sample.

Processing continues by washing retention member 216 with liquid ofreservoir 279 to separate remaining inhibitors from polynucleotidesretained by retention member 216. To wash retention member 216, valve206 is closed and gates 242, 246 of first reservoir 240 are opened.Actuator 244 is actuated and moves wash liquid within reservoir 279along channels 257, 261, and 214, through processing region 220, andinto waste reservoir 232. The wash liquid moves sample that may haveremained within channels 204, 257, 261, and 214 through the processingregion and into waste chamber 232. Once the trailing edge of the washliquid reaches vent 212, the gas pressure generated by actuator 244 isvented and further motion of the liquid is stopped.

The volume of wash liquid moved by actuator 244 through processingregion 220 is typically at least about 2 times the void volume ofprocessing region 220 (e.g., at least about 3 times the void volume) andcan be about 10 times the void volume or less (e.g., about 5 times thevoid volume or less). Processing region is typically at a temperature ofabout 50° C. or less (e.g., 30° C. or less) during washing. Exemplarywash fluids include liquids discussed with respect to reservoirs 279 and281.

Processing continues by releasing polynucleotides from retention member216. Typically, wash liquid from reservoir 279 is replaced with releaseliquid (e.g., an hydroxide solution) from reservoir 281 before releasingthe polynucleotides. Valve 208 is closed and gates 250, 252 are opened.Actuator 248 is actuated thereby moving release liquid within reservoir281 along channels 261, 214 and into processing region 220 and incontact with retention member 216. When the trailing edge of releaseliquid from reservoir 281 reaches hydrophobic vent 212, pressuregenerated by actuator 248 is vented stopping the further motion of theliquid. The volume of liquid moved by actuator 248 through processingregion 220 is typically at least about equal to the void volume of theprocessing region 220 (e.g., at least about 2 times the void volume) andcan be about 10 times the void volume or less (e.g., about 5 times thevoid volume or less).

Once retention member 216 with retained polynucleotides has beencontacted with liquid from reservoir 281, a releasing step is typicallyperformed. Typically, the releasing step includes heating release liquidpresent within processing region 216. Generally, the liquid is heated toa temperature insufficient to boil liquid in the presence of theretention member. In some embodiments, the temperature is 100° C. orless (e.g., less than 100° C., about 97° C. or less). In someembodiments, the temperature is about 65° C. or more (e.g., about 75° C.or more, about 80° C. or more, about 90° C. or more). In someembodiments, the temperature maintained for about 1 minute or more(e.g., about 2 minutes or more, about 5 minutes or more, about 10minutes or more). In some embodiments, the temperature is maintained forabout 30 minutes (e.g., about 15 minutes or less, about 10 minutes orless, about 5 minutes or less). In an exemplary embodiment, processingregion 220 is heated to between about 65 and 90° C. (e.g., to about 70°C.) for between about 1 and 7 minutes (e.g., for about 2 minutes).

The polynucleotides are released into the liquid present in theprocessing region 220 (e.g., the polynucleotides are typically releasedinto an amount of release liquid having a volume about the same as thevoid volume of the processing region 220). Typically, thepolynucleotides are released into about 10 microliters or less (e.g.,about 5 microliters or less, about 2.5 microliters or less) of liquid.

In certain embodiments, the ratio of the volume of original sample movedthrough the processing region 220 to the volume of liquid into which thepolynucleotides are released is at least about 10 (e.g., at least about50, at least about 100, at least about 250, at least about 500, at leastabout 1000). In some embodiments, polynucleotides from a sample having avolume of about 2 ml can be retained within the processing region, andreleased into about 4 microliters or less (e.g., about 3 microliters orless, about 2 microliters or less, about 1 microliter or less) ofliquid.

The liquid into which the polynucleotides are released typicallyincludes at least about 50% (e.g., at least about 75%, at least about85%, at least about 90%) of the polynucleotides present in the samplethat entered processing region 220. The concentration of polynucleotidespresent in the release liquid may be higher than in the original samplebecause the volume of release liquid is typically less than the volumeof the original liquid sample moved through the processing region. Forexample the concentration of polynucleotides in the release liquid maybe at least about 10 times greater (e.g., at least about 25 timesgreater, at least about 100 times greater) than the concentration ofpolynucleotides in the sample introduced to device 200. Theconcentration of inhibitors present in the liquid into which thepolynucleotides are released is generally less than concentration ofinhibitors in the original fluidic sample by an amount sufficient toincrease the amplification efficiency for the polynucleotides.

The time interval between introducing the polynucleotide containingsample to processing region 220 and releasing the polynucleotides intothe release liquid is typically about 15 minutes or less (e.g., about 10minutes or less, about 5 minutes or less).

Liquid including the released polynucleotides may be removed from theprocessing region 220 as follows. Valves 210 and 234 are closed. Gates238 and 258 are opened. Actuator 254 is actuated to generate pressurethat moves liquid and polynucleotides from processing region 220, intochannel 230, and toward outlet 236. The liquid with polynucleotides canbe removed using, for example, a syringe or automated sampling device.Depending upon the liquid in contact with retention member 216 duringpolynucleotide release, the solution with released polynucleotide may beneutralized with an amount of buffer (e.g., an equal volume of 25-50 mMTris-HCl buffer pH 8.0).

While releasing the polynucleotides has been described as including aheating step, the polynucleotides may be released without heating. Forexample, in some embodiments, the liquid of reservoir 281 has an ionicstrength, pH, surfactant concentration, composition, or combinationthereof that releases the polynucleotides from the retention member.

While the polynucleotides have been described as being released into asingle volume of liquid present within processing region 220, otherconfigurations can be used. For example, polynucleotides may be releasedwith the concomitant (stepwise or continuous) introduction of fluid intoand/or through processing region 220. In such embodiments, thepolynucleotides may be released into liquid having a volume of about 10times or less (e.g., about 7.5 times or less, about 5 times or less,about 2.5 times or less, about 2 times or less) than the void volume ofthe processing region 220.

While reservoirs 279, 281 have been described as holding liquids betweenfirst and second gates, other configurations can be used. For example,liquid for each reservoir may be held within a pouch (e.g., a blisterpack) isolated from network 201 by an generally impermeable membrane.The pouch is configured so that a user can rupture the membrane drivingliquid into reservoirs 279, 281 where actuators 244, 248 can move theliquid during use.

While processing regions have been described as having microliter scaledimensions, other dimensions can be used. For example, processingregions with surfaces (e.g., particles) configured to preferentiallyretain polynucleotides as opposed to inhibitors may have large volumes(e.g., many tens of microliters or more, at least about 1 milliliter ormore). In some embodiments, the processing region has a bench-top scale.

While processing region 220 has been described as having a retentionmember formed of multiple surface-modified particles, otherconfigurations can be used. For example, in some embodiments, processingregion 220 includes a retention member configured as a porous member(e.g., a filter, a porous membrane, or a gel matrix) having multipleopenings (e.g., pores and/or channels) through which polynucleotidespass. Surfaces of the porous member are modified to preferentiallyretain polynucleotides. Filter membranes available from, for example,Osmonics, are formed of polymers that may be surface-modified and usedto retain polynucleotides within processing region 220. In someembodiments, processing region 220 includes a retention memberconfigured as a plurality of surfaces (e.g., walls or baffles) throughwhich a sample passes. The walls or baffles are modified topreferentially retain polynucleotides.

While processing region 220 has been described as a component of amicrofluidic network, other configurations can be used. For example, insome embodiments, the retention member can be removed from a processingregion for processing elsewhere. For example, the retention member maybe contacted with a mixture comprising polynucleotides and inhibitors inone location and then moved to another location at which thepolynucleotides are removed from the retention member.

While reservoirs 275 have been shown as dispersed within a carrier,other configurations may be used. For example, reservoirs 275 can beencased within a flexible enclosure formed by a, for example, (e.g., amembrane, for example, an enclosure such as a sack). In someembodiments, reservoirs are loose within chamber 272. In suchembodiments, actuator 244 may include a porous member having pores toosmall to permit passage of reservoirs 275 but large enough to permit gasto exit chamber 272.

Microfluidic devices with various components are described in U.S.provisional application No. 60/553,553 filed Mar. 17, 2004 by Parunak etal., which application is incorporated herein by reference.

While microfluidic device 300 has been described as configured toreceive polynucleotides already released from cells, microfluidicdevices can be configured to release polynucleotides from cells (e.g.,by lysing the cells). For example, referring to FIGS. 4-6, amicrofluidic device 300 includes a sample lysing chamber 302 in whichcells are lysed to release polynucleotides therein. Microfluidic device300 further includes substrate layers L1-L3, a microfluidic network 304(only portions of which are seen in FIG. 4), and liquid reagentreservoirs R1-R4. Liquid reagent reservoirs R1-R4 hold liquid reagents(e.g., for processing sample material) and are connected to network 304by reagent ports RP1-RP4.

Network 304 is substantially defined between layers L2 and L3 butextends in part between all three layers L1-L3. Microfluidic network 304includes multiple components including channels Ci, valves Vi, doublevalves V′_(i), gates G1, mixing gates MGi, vents Hi, gas actuators(e.g., pumps) Pi, a first processing region B1, a second processingregion B2, detection zones Di, air vents AVi, and waste zones Wi.Components of network 304 are typically thermally actuated. As seen inFIG. 7, a heat source network 312 includes heat sources (e.g., resistiveheat sources) having locations that correspond to components ofmicrofluidic network 304. For example, the locations of heat sources HPicorrespond to the locations of actuators Pi, the locations of heatsources HGi correspond to locations of gates G1 and mixing gates, thelocations of heat sources HVi correspond to the locations of valves Viand double valves V′i, and the locations of heat sources HD1 correspondto the locations of processing chambers Di of network 304. In use, thecomponents of device 300 are disposed in thermal contact withcorresponding heat sources of network 312, which is typically operatedusing a processor as described above for device 200. Heat source network312 can be integral with or separate from device 300 as described fordevice 200.

We next discuss components of microfluidic device 300.

Air vents AVi are components that allow gas (e.g., air) displaced by themovement of liquids within network 304 to be vented so that pressurebuildup does not inhibit desired movement of the liquids. For example,air vent AV2 permits liquid to move along channel C14 and into channelC16 by venting gas downstream of the liquid through vent AV2.

Valves Vi are components that have a normally open state allowingmaterial to pass along a channel from a position on one side of thevalve (e.g., upstream of the valve) to a position on the other side ofthe valve (e.g., downstream of the valve). The valves Vi can have thesame structure as valves of microfluidic device 200.

As seen in FIGS. 8 and 9, double valves V′i are also components thathave a normally open state allowing material to pass along a channelfrom a position on one side of the valve (e.g., upstream of the valve)to a position on the other side of the valve (e.g., downstream of thevalve). Taking double valve V11′ of FIGS. 8 and 9 as an example, doublevalves Vi′ include first and second masses 314, 316 of a TRS (e.g., aeutectic alloy or wax) spaced apart from one another on either side of achannel (e.g., channel C14). Typically, the TRS masses 314, 316 areoffset from one another (e.g., by a distance of about 50% of a width ofthe TRS masses or less). Material moving through the open valve passesbetween the first and second TRS masses 314,316. Each TRS mass 314, 316is associated with a respective chamber 318, 320, which typicallyincludes a gas (e.g., air).

The TRS masses 314, 316 and chambers 318, 320 of double valve Vi′ are inthermal contact with a corresponding heat source HV11′ of heat sourcenetwork 312. Actuating heat source HV11′ causes TRS masses 314, 316 totransition to a more mobile second state (e.g., a partially meltedstate) and increases the pressure of gas within chambers 318, 320. Thegas pressure drives TRS masses 314, 316 across channel C11 and closesvalve HV11′ (FIG. 9). Typically, masses 314, 316 at least partiallycombine to form a mass 322 that obstructs channel C11.

Returning to FIG. 6, gates G1 are components that have a normally closedstate that does not allow material to pass along a channel from aposition on one side of the gate to another side of the gate. Gates G1can have the same structure as described for gates of device 200.

As seen in FIGS. 10A-10D, mixing gates MGi are components that allow twovolumes of liquid to be combined (e.g., mixed) within network 304.Mixing gates MGi are discussed further below.

Actuators Pi are components that provide a gas pressure to move material(e.g., sample material and/or reagent material) between one location ofnetwork 304 and another location. Actuators Pi can be the same asactuators of device 200. For example, each actuator Pi includes achamber with a mass 273 of TEM that can be heated to pressurize gaswithin the chamber. Each actuator Pi includes a corresponding gate G1(e.g., gate G2 of actuator P1) that prevents liquid from entering thechamber of the actuator. The gate is typically actuated (e.g., opened)to allow pressure created in the chamber of the actuator to enter themicrofluidic network.

Waste chambers Wi are components that can receive waste (e.g., overflow)liquid resulting from the manipulation (e.g., movement and/or mixing) ofliquids within network 304. Typically, each waste chamber Wi has anassociated air vent that allows gas displaced by liquid entering thechamber to be vented.

First processing region B1 is a component that allows polynucleotides tobe concentrated and/or separated from inhibitors of a sample. Processingregion B1 can be configured and operated as processing region 220 ofdevice 200. In some embodiments, first processing region B1 includes aretention member (e.g., multiple particles (e.g., microspheres orbeads), a porous member, multiple walls) having at least one surfacemodified with one or more ligands as described for processing region220. For example, the ligand can include one or more polyamides (e.g.,poly-cationic polyamides such as poly-L-lysine, poly-D-lysine,poly-DL-ornithine). In some embodiments, particles of the retentionmember are disposed lysing chamber 302 and are moved into processingregion B1 along with sample material.

Second processing region B2 is a component that allows material (e.g.,sample material) to be combined with compounds (e.g., reagents) fordetermining the presence of one or more polynucleotides. In someembodiments, the compounds include one or more PCR reagents (e.g.,primers, control plasmids, and polymerase enzymes). Typically, thecompounds are stored within processing region as one or more lyophilizedparticles (e.g., pellets). The particles generally have a roomtemperature (e.g., about 20° C.) shelf-life of at least about 6 months(e.g., at least about 12 months). Liquid entering the second processingregion B2 dissolves (e.g., reconstitutes) the lyophilized compounds.

Typically, the lyophilized particle(s) of processing region B2 have anaverage volume of about 5 microliters or less (e.g., about 4 microlitersor less, about 3 microliters or less, about 2 microliters or less). Insome embodiments, the lyophilized particle(s) of processing region B2have an average diameter of about 4 mm or less (e.g., about 3 mm orless, about 2 mm or less) In an exemplary embodiment the lyophilizedparticle(s) have an average volume of about 2 microliters and an averagediameter of about 1.35 mm. Lyophilized particles for determining thepresence of one or more polynucleotides typically include multiplecompounds. In some embodiments, the lyophilized particles include one ormore compounds used in a reaction for determining the presence of apolynucleotide and/or for increasing the concentration of thepolynucleotide. For example, lypophilized particles can include one ormore enzymes for amplifying the polynucleotide as by PCR. We nextdiscuss exemplary lyophilized particles that include exemplary reagentsfor the amplification of polynucleotides associated with group Bstreptococcus (GBS) bacteria. In some embodiments, the lyophilizedparticles include a cryoprotectant, one or more salts, one or moreprimers (e.g., GBS Primer F and/or GBS Primer R), one or more probes(e.g., GBS Probe—FAM), one or more internal control plasmids, one ormore specificity controls (e.g., Streptococcus pneumoniae DNA as acontrol for PCR of GBS), one or more PCR reagents (e.g., dNTPs and/ordUTPs), one or more blocking or bulking agents (e.g., non-specificproteins (e.g., bovine serum albumin (BSA), RNAseA, or gelatin), and apolymerase (e.g., glycerol-free Taq Polymerase). Of course, othercomponents (e.g., other primers and/or specificity controls) can be usedfor amplification of other polynucleotides.

Cryoprotectants generally help increase the stability of thelypophilized particles and help prevent damage to other compounds of theparticles (e.g., by preventing denaturation of enzymes duringpreparation and/or storage of the particles). In some embodiments, thecryoprotectant includes one or more sugars (e.g., one or moredissacharides (e.g., trehalose, melizitose, raffinose)) and/or one ormore poly-alcohols (e.g., mannitol, sorbitol).

Lyophilized particles can be prepared as desired. Typically, compoundsof the lyophilized particles are combined with a solvent (e.g., water)to make a solution, which is then placed (e.g., in discrete aliquots(e.g., drops) such as by pipette) onto a chilled hydrophobic surface(e.g., a diamond film or a polytetrafluorethylene surface). In general,the temperature of the surface is reduced to near the temperature ofliquid nitrogen (e.g., about −150° F. or less, about −200° F. or less,about −275° F. or less). The solution freezes as discrete particles. Thefrozen particles are subjected to a vacuum while still frozen for apressure and time sufficient to remove the solvent (e.g., bysublimation) from the pellets.

In general, the concentrations of the compounds in the solution fromwhich the particles are made is higher than when reconstituted in themicrofluidic device. Typically, the ratio of the solution concentrationto the reconstituted concentration is at least about 3 (e.g., at leastabout 4.5). In some embodiments, the ratio is about 6.

An exemplary solution for preparing lyophilized pellets for use in theamplification of polynucleotides indicative of the presence of GBS canbe made by combining a cryoprotecant (e.g., 120 mg of trehalose as drypowder), a buffer solution (e.g., 48 microliters of a solution of 1MTris at pH 8.4, 2.5M KCl, and 200 mM MgCl₂), a first primer (e.g., 1.92microliters of 500 micromolar GBS Primer F (Invitrogen)), a secondprimer (e.g., 1.92 microliters of 500 micromolar GBS Primer R(Invitrogen)), a probe (e.g., 1.92 microliters of 250 micromolar GBSProbe—FAM (IDT/Biosearch Technologies)), an control probe (e.g., 1.92microliters of 250 micromolar Cal Orange 560 (Biosearch Technologies)),a template plasmid (e.g., 0.6 microliters of a solution of 10⁵ copiesplasmid per microliter), a specificity control (e.g., 1.2 microliters ofa solution of 10 nanograms per microliter (e.g., about 5,000,000 copiesper microliter) streptococcus pneumoniae DNA (ATCC)), PCR reagents(e.g., 4.8 microliters of a 100 millimolar solution of dNTPs (Epicenter)and 4.microliters of a 20 millimolar solution of dUTPs (Epicenter)), abulking agent (e.g., 24 microliters of a 50 milligram per millilitersolution of BSA (Invitrogen)), a polymerase (e.g., 60 microliters of a 5U per microliter solution of glycerol-free Taq Polymerase(Invitrogen/Eppendorf)) and a solvent (e.g., water) to make about 400microliters of solution. About 200 aliquots of about 2 microliters eachof this solution are frozen and desolvated as described above to make200 pellets. When reconstituted, the 200 particles make a PCR reagentsolution having a total volume of about 2.4 milliliters.

As seen in FIG. 5, reagent reservoirs R1 are configured to hold liquidreagents (e.g., water, buffer solution, hydroxide solution) separatedfrom network 304 until ready for use. Reservoirs R1 include an enclosure329 that defines a sealed space 330 for holding liquids. Each space 330is separated from reagent port RPi and network 304 by a lower wall 33 ofenclosure 329. A portion of enclosure 329 is formed as a piercing member331 oriented toward the lower wall 333 of each enclosure. When device300 is to be used, reagent reservoirs R1 are actuated by depressingpiercing member 331 to puncture wall 333. Piercing member 331 can bedepressed by a user (e.g., with a thumb) or by the operating system usedto operate device 300.

When wall 333 is punctured, fluid from the reservoir enters network 333.For example, as seen in FIGS. 5 and 6, liquid from reservoir R2 entersnetwork 304 by port RP2 and travels along a channel C2. Gate G3 preventsthe liquid from passing along channel C8. Excess liquid passes alongchannel C7 and into waste chamber W2. When the trailing edge of liquidfrom reservoir R2 passes hydrophobic vent H2, pressure created withinthe reservoir is vented stopping further motion of the liquid.Consequently, network 304 receives an aliquot of liquid reagent having avolume defined by the volume of channel C2 between a junction J1 and ajunction J2. When actuator P1 is actuated, this aliquot of reagent ismoved further within network 304. Reagent reservoirs R1, R3, and R4 areassociated with corresponding channels, hydrophobic vents, andactuators.

In the configuration shown, reagent reservoir R1 typically holds arelease liquid (e.g., a hydroxide solution as discussed above for device200) for releasing polynucleotides retained within processing region B1.Reagent reservoir R2 typically holds a wash liquid (e.g., a buffersolution as discussed above for device 200) for removing un-retainedcompounds (e.g., inhibitors) from processing region B1 prior toreleasing the polynucleotides. Reagent reservoir R3 typically holds aneutralization buffer (e.g., 25-50 mM Tris-HCl buffer at pH 8.0).Reagent reservoir R4 typically holds deionized water.

Lysing chamber 302 is divided into a primary lysing chamber 306 and awaste chamber 308. Material cannot pass from one of chambers 306, 308into the other chamber without passing through at least a portion ofnetwork 304. Primary lysing chamber 306 includes a sample input port SP1for introducing sample to chamber 306, a sample output port SP2connecting chamber 306 to network 304, and lyophilized reagent LP thatinteract with sample material within chamber 306 as discussed below.Input port SP1 includes a one way valve that permits material (e.g.,sample material and gas) to enter chamber 306 but limits (e.g.,prevents) material from exiting chamber 308 by port SP1. Typically, portSP1 includes a fitting (e.g., a Luer fitting) configured to mate with asample input device (e.g., a syringe) to form a gas-tight seal. Primarychamber 306 typically has a volume of about 5 milliliters or less (e.g.,about 4 milliliters or less). Prior to use, primary chamber 306 istypically filled with a gas (e.g., air).

Waste chamber 308 includes a waste portion W6 by which liquid can enterchamber 308 from network 304 and a vent 310 by which gas displaced byliquid entering chamber 308 can exit.

Lyophilized reagent particles LP of lysing chamber 302 include one ormore compounds (e.g., reagents) configured to release polynucleotidesfrom cells (e.g., by lysing the cells). For example, particles LP caninclude one or more enzymes configured to reduce (e.g., denature)proteins (e.g., proteinases, proteases (e.g., pronase), trypsin,proteinase K, phage lytic enzymes (e.g., PlyGBS)), lysozymes (e.g., amodified lysozyme such as ReadyLyse), cell specific enzymes (e.g.,mutanolysin for lysing group B streptococci)).

In some embodiments, articles LP typically alternatively or additionallyinclude components for retaining polynucleotides as compared toinhibitors. For example, particles LP can include multiple particles 218surface modified with ligands as discussed above for device 200.Particles LP can include enzymes that reduce polynucleotides that mightcompete with a polynucleotide to be determined for binding sites on thesurface modified particles. For example, to reduce RNA that mightcompete with DNA to be determined, particles LP may include an enzymesuch as an RNAase (e.g., RNAseA ISC BioExpress (Amresco)).

In an exemplary embodiment, particles LP cells include a cryoprotecant,particles modified with ligands configured to retain polynucleotides ascompared to inhibitors, and one or more enzymes.

Typically, particles LP have an average volume of about 35 microlitersor less (e.g., about 27.5 microliters or less, about 25 microliters orless, about 20 microliters or less). In some embodiments, the particlesLP have an average diameter of about 8 mm or less (e.g., about 5 mm orless, about 4 mm or less) In an exemplary embodiment the lyophilizedparticle(s) have an average volume of about 20 microliters and anaverage diameter of about 3.5 mm.

Particles LP can be prepared as desired. Typically, the particles areprepared using a cryoprotectant and chilled hydrophobic surface asdescribed above. For example, a solution for preparing particles LP canbe prepared by combining a cryoprotectant (e.g., 6 grams of trehalose),a plurality of particles modified with ligands (e.g., about 2milliliters of a suspension of carboxylate modified particles withpoly-D-lysine ligands), a protease (e.g., 400 milligrams of pronase), anRNAase (e.g., 30 milligrams of RNAseA (activity of 120 U per milligram),an enzyme that digests peptidoglycan (e.g., ReadyLyse (e.g., 160microliters of a 30000 U per microliter solution of ReadyLyse)), a cellspecific enzyme (e.g., mutanolysin (e.g., 200 microliters of a 50 U permicroliter solution of mutanolysin), and a solvent (e.g., water) to makeabout 20 milliters. About 1000 aliquots of about 20 microliters each ofthis solution are frozen and desolvated as described above to make 1000pellets. When reconstituted, the pellets are typically used to make atotal of about 200 milliliters of solution.

In use, device 300 can be operated as follows. Valves Vi and Vi′ ofnetwork 304 are configured in the open state. Gates G1 and mixing gatesMGi of network 304 are configured in the closed state. Reagent portsR1-R4 are depressed to introduce liquid reagents into network 304 asdiscussed above. A sample is introduced to lysing chamber 302 via portSP1 and combined with lyophilized particles LP within primary lysingchamber 306. Typically, the sample includes a combination of particles(e.g., cells) and a buffer solution. For example, an exemplary sampleincludes about 2 parts whole blood to 3 about parts buffer solution(e.g., a solution of 20 mM Tris at pH 8.0, 1 mM EDTA, and 1% SDS).Another exemplary sample includes group B streptococci and a buffersolution (e.g., a solution of 20 mM Tris at pH 8.0, 1 mM EDTA, and 1%Triton X-100).

In general, the volume of sample introduced is smaller than the totalvolume of primary lysing chamber 306. For example, the volume of samplemay be about 50% or less (e.g., about 35% or less, about 30% or less) ofthe total volume of chamber 306. A typical sample has a volume of about3 milliliters or less (e.g., about 1.5 milliliters or less). A volume ofgas (e.g., air) is generally introduced to primary chamber 306 alongwith the sample. Typically, the volume of gas introduced is about 50% orless (e.g., about 35% or less, about 30% or less) of the total volume ofchamber 306. The volume of sample and gas combine to pressurize the gasalready present within chamber 306. Valve 307 of port SP1 prevents gasfrom exiting chamber 306. Because gates G3, G4, G8, and G10 are in theclosed state, the pressurized sample is prevented from entering network304 via port SP2.

The sample dissolves particles LP in chamber 306. Reconstituted lysingreagents (e.g., ReadyLyse, mutanolysin) begin to lyse cells of thesample releasing polynucleotides. Other reagents (e.g., protease enzymessuch as pronase) begin to reduce or denature inhibitors (e.g., proteins)within the sample. Polynucleotides from the sample begin to associatewith (e.g., bind to) ligands of particles 218 released from particlesLP. Typically, the sample within chamber 306 is heated (e.g., to atleast about 50° C., to at least about 60° C.) for a period of time(e.g., for about 15 minutes or less, about 10 minutes or less, about 7minutes or less) while lysing occurs. In some embodiments, opticalenergy is used at least in part to heat contents of lysing chamber 306.For example, the operating system used to operate device 300 can includea lamp (e.g., a lamp primarily emitting light in the infrared) disposedin thermal and optical contact with chamber 306. Chamber 306 includes atemperature sensor TS used to monitor the temperature of the samplewithin chamber 306. The lamp output is increased or decreased based onthe temperature determined with sensor TS.

Continuing with the operation of device 300, G2 is actuated (e.g.,opened) providing a path between port SP2 of primary lysing chamber 306and port W6 of lysing waste chamber 308. The path extends along channelC9, channel C8, through processing region B1, and channel C11. Pressurewithin chamber 306 drives the lysed sample material (containing lysate,polynucleotides bound to particles 218, and other sample components)along the pathway. Particles 218 (with polynucleotides) are retainedwithin processing region B1 (e.g., by a filter) while the liquid andother components of the sample flow into waste chamber 308. After aperiod of time (e.g., between about 2 and about 5 minutes), the pressurein lysing chamber 306 is vented by opening gate G1 to create a secondpathway between ports SP2 and W6. Double valves V1′ and V8′ are closedto isolate lysing chamber 302 from network 304.

Operation of device 300 continues by actuating pump P1 and opening gatesG2,G3 and G9. Pump P1 drives wash liquid in channel C2 downstream ofjunction J1 through processing region B1 and into waste chamber W5. Thewash liquid removes inhibitors and other compounds not retained byparticles 218 from processing region B1. When the trailing edge of thewash liquid (e.g., the upstream interface) passes hydrophobic vent H14,the pressure from actuator P1 vents from network 304, stopping furthermotion of the liquid. Double valves V2′ and V9′ are closed.

Operation continues by actuating pump P2 and opening gates G6, G4 and G8to move release liquid from reagent reservoir R1 into processing regionB1 and into contact with particles 218. Air vent AV1 vents pressureahead of the moving release liquid. Hydrophobic vent H6 vents pressurebehind the trailing edge of the release liquid stopping further motionof the release liquid. Double valves V6′ and V10′ are closed.

Operation continues by heating processing region B1 (e.g., by heatingparticles 218) to release the polynucleotides from particles 218. Theparticles can be heated as described above for device 200. Typically,the release liquid includes about 15 mM hydroxide (e.g., NaOH solution)and the particles are heated to about 70° C. for about 2 minutes torelease the polynucleotides from the particles 218.

Operation continues by actuating pump P3 and opening gates G5 and G10 tomove release liquid from process region B1 downstream. Air vent AV2vents gas pressure downstream of the release liquid allowing the liquidto move into channel C16. Hydrophobic vent H8 vents pressure fromupstream of the release liquid stopping further movement. Double valveV11′ and valve V14 are closed.

Referring to FIGS. 10A-10D, mixing gate MG11 is used to mix a portion ofrelease liquid including polynucleotides released from particles 218 andneutralization buffer from reagent reservoir R3. FIG. 10A shows themixing gate MG11 region prior to depressing reagent reservoir R3 tointroduce the neutralization buffer into network 304. FIG. 10B shows themixing gate MG11 region, after the neutralization buffer has beenintroduced into channels C13 and C12. Double valve V13′ is closed toisolate network 304 from reagent reservoir R3. Double valve V12′ isclosed to isolate network 304 from waste chamber W3. The neutralizationbuffer contacts one side of a mass 324 of TRS of gate MG11.

FIG. 10 c shows the mixing gate MG11 region after release liquid hasbeen moved into channel C16. The dimensions of microfluidic network 304(e.g., the channel dimensions and the position of hydrophobic vent H8)are configured so that the portion of release liquid positioned betweenjunctions J3 and J4 of channels C16 and C14 corresponds approximately tothe volume of liquid in contact with particles 218 during the releasestep. In some embodiments, the volume of liquid positioned betweenjunctions J3 and J4 is less than about 5 microliters (e.g., about 4microliters or less, about 2.5 microliters or less). In an exemplaryembodiment the volume of release liquid between junctions J3 and J4 isabout 1.75 microliters. Typically, the liquid between junctions J3 andJ4 includes at least about 50% of polynucleotides (at least about 75%,at least about 85%, at least about 90%) of the polynucleotides presentin the sample that entered processing region B1. Valve V14 is closed toisolate network 304 from air vent AV2.

Before actuating mixing gate MG11, the release liquid at junction J4 andthe neutralization buffer at a junction J6 between channels C13 and C12are separated only be mass 324 of TRS (e.g., the liquids are not spacedapart by a volume of gas). To combine the release liquid andneutralization buffer, pump P4 and gates G12, G13, and MG11 areactuated. Pump P4 drives the volume of neutralization liquid betweenjunctions J5 and J6 and the volume of release liquid between junctionsJ4 and J3 into mixing channel C15 (FIG. 10D). Mass 324 of TRS typicallydisperses and/or melts allowing the two liquids to combine. The combinedliquids include a downstream interface 335 (formed by junction J3) andan upstream interface (formed by junction J5). The presence of theseinterfaces allows more efficient mixing (e.g., recirculation of thecombined liquid) than if the interfaces were not present. As seen inFIG. 10D, mixing typically begins near the interface between the twoliquids. Mixing channel C15 is typically at least about as long (e.g.,at least about twice as long) as a total length of the combined liquidswithin the channel.

The volume of neutralization buffer combined with the release liquid isdetermined by the channel dimensions between junction J5 and J6.Typically, the volume of combined neutralization liquid is about thesame as the volume of combined release liquid. In some embodiments, thevolume of liquid positioned between junctions J5 and J6 is less thanabout 5 microliters (e.g., about 4 microliters or less, about 2.5microliters or less). In an exemplary embodiment the volume of releaseliquid between junctions J5 and J6 is about 2.25 microliters (e.g., thetotal volume of release liquid and neutralization buffer is about 4microliters).

Returning to FIG. 6, the combined release liquid and neutralizationbuffer move along mixing channel C15 and into channel C32 (venteddownstream by air vent AV8). Motion continues until the upstreaminterface of the combined liquids passes hydrophobic vent H11, whichvents pressure from actuator P4 stopping further motion of the combinedliquids.

Continuing with operation of device 300, actuator P5 and gates G14, G15and G17 are actuated to dissolve the lyophilized PCR particle present insecond processing region B2 in water from reagent reservoir R4.Hydrophobic vent H10 vents pressure from actuator P5 upstream of thewater stopping further motion. Dissolution typically occurs in about 2minutes or less (e.g., in about 1 minute or less). to dissolvePCR-reagent pellet. Valve V17 is closed.

Continuing with operation of device 300, actuator P6 and gate G16 areactuated to drive the dissolved compounds of the lyophilized particlefrom processing region B2 into channel C31, where the dissolved reagentsmix to form a homogenous dissolved lyophilized particle solution.Actuator P6 moves the solution into channels C35 and C33 (venteddownstream by air vent AV5). Hydrophobic vent H9 vents pressuregenerated by actuator P6 upstream of the solution stopping furthermotion. Valves V18, V19, V20′, and V22′ are closed.

Continuing with operation of device 300, actuator P7 and gates G18, MG20and G22 are actuated to combine (e.g., mix) a portion of neutralizedrelease liquid in channel 32 between gate MG20 and gate G22 and aportion of the dissolved lyophilized particle solution in channel C35between gate G18 and MG20. The combined liquids travel long a mixingchannel C37 and into detection region D2. An air vent AV3 vents gaspressure downstream of the combined liquids. When the upstream interfaceof the combined liquids passes hydrophobic vent H13, the pressure fromactuator P7 is vented and the combined liquids are positioned withindetection region D2.

Actuator P8 and gates MG2, G23, and G19 are actuated to combine aportion of water from reagent reservoir R4 between MG2 and gate G23 witha second portion of the dissolved lyophilized particle solution inchannel C33 between gate G19 and MG2. The combined liquids travel long amixing channel C41 and into detection region D1. An air vent AV4 ventsgas pressure downstream of the combined liquids. When the upstreaminterface of the combined liquids passes hydrophobic vent H12, thepressure from actuator P8 is vented and the combined liquids arepositioned within detection region D1.

Continuing with operation of device 300, double valves V26′ and V27′ areclosed to isolate detection region D1 from network 304 and double valvesV24′ and V25′ are closed to isolate detection region D2 from network304. The contents of each detection region (neutralized release liquidwith sample polynucleotides in detection region D2 with PCR reagentsfrom dissolved lyophilized particle solution and deionized water withPCR reagents from dissolved lyophilized particle solution in detectionregion D1) are subjecting to heating and cooling steps to amplifypolynucleotides (if present in detection region D2). The double valvesof each detection region prevent evaporation of the detection regioncontents during heating. The amplified polynucleotides are typicallydetected using fluorescence detection.

Referring to FIG. 11, a device 700 is configured to process apolynucleotide-containing sample, such as to prepare the sample foramplification of the polynucleotides. Device 700 includes a samplereservoir 704, a reagent reservoir 706, a gas pressure generator 708, aclosure (e.g., a cap 710), and a processing region 702 including aretention member 704 having a plurality of particles (e.g. carboxylatebeads 705 surface-modified with a ligand, e.g., poly-L-lysine and/orpoly-D-lysine). Retention member 705 and beads 705 may share any or allproperties of retention member 216 and surface-modified particles 218.Device 700 also includes an opening 716 and a valve, e.g., a thermallyactuated valve 714 for opening and closing opening 716.

In use, a polynucleotide-containing sample is added to sample reservoir704. Typical sample amounts range from about 100 μL to about 2 mL,although greater or smaller amounts may be used.

Reagent reservoir 706 may be provided to users of device 700 withpre-loaded reagent. Alternatively, device 700 may be configured so thatusers add reagent to device 700. In any event, the reagents may include,e.g., NaOH solutions and/or buffer solutions such as any of suchsolutions discussed herein.

Once sample and, if necessary, reagent have been added to device 700,cap 710 is closed to prevent evaporation of sample and reagentmaterials.

Referring also to FIG. 12, an operator 718 is configured to operatedevice 700. Operator 718 includes a first heat source 720 and a secondheat source 722. First heat source 720 heats sample present withinsample reservoir 704, such as to lyse cells of thepolynucleotide-containing sample to prepare free polynucleotides.

Device 700 may also include an enzyme reservoir 712 comprising anenzyme, e.g., a protease such as pronase, configured to cleave peptidebonds of polypeptides present in the polynucleotide-containing sample.Enzyme reservoir 712 may be provided to users of device 700 withpre-loaded enzyme. Alternatively, device 700 may be configured so thatusers add enzyme to device 700.

Device 700 may be used to reduce the amount of inhibitors presentrelative to the amount of polynucleotides to be determined. Thus, thesample is eluted through processing region 702 to contact constituentsof the sample with beads 705. Beads 705 retain polynucleotides of thesample as compared to inhibitors as described elsewhere herein. Withvalve 714 in the open state, sample constituents not retained inprocessing region 702 exit device 700 via the opening.

Once the polynucleotide-containing sample has eluted through processingregion 702, an amount of reagent, e.g., a wash solution, e.g., a buffersuch as Tris-EDTA pH 8.0 with 1% Triton X 100 is eluted throughprocessing region 702. The wash solution is generally stored in reagentreservoir 706, which may include a valve configured to release an amountof wash solution. The wash solution elutes remainingpolynucleotide-containing sample and inhibitors without eluting retainedpolynucleotides.

Once inhibitors have been separated from retained polynucleotides, thepolynucleotides are released from beads 705. In some embodiments,polynucleotides are released by contacting the beads 705 with a releasesolution, e.g., a NaOH solution or buffer solution having a pH differentfrom that of the wash solution. Alternatively, or in combination, beads705 with retained polynucleotides are heated, such as by using secondheat source 722 of operator 718. When heat is used to release thepolynucleotides, the release solution may be identical with the washsolution.

Gas pressure generator 708 may be used to expel an amount of releasesolution with released polynucleotides from device 700. Gas pressuregenerator and/or operator 718 may include a heat source to heat gaspresent within generator 708. The heated gas expands and provides thegas pressure to expel sample. In some embodiments, and whether or notthermally generated gas pressure is used, gas pressure generator 708 isconfigured to expel a predetermined volume of material. Typically, theamount of expelled solution is less than about 500 μL, less than about250 μL, less than about 100 μL, less than about 50 μL, e.g., less thanabout 25 μL.

EXAMPLES

The following Examples are illustrative and not intended to be limiting.

Preparing Retention Member

Carboxylate surface magnetic beads (Sera-Mag Magnetic Carboxylatemodified, Part #3008050250, Seradyn) at a concentration of about 10¹¹mL⁻¹ were activated for 30 minutes using N-hydroxylsuccinimide (NHS) and1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) in a pH 6.1 500 mM2-(N-Morpholinio)-ethanesulfonic acid (MES) buffer solution. Activatedbeads were incubated with 3000 Da or 300,000 Da average molecular weightpoly-L-lysine (PLL). After 2 washes to remove unbound PLL, beads wereready for use.

Microfluidic Device

Referring to FIGS. 13 and 14, a microfluidic device 300 was fabricatedto demonstrate separation of polynucleotides from inhibitors. Device 300comprises first and second substrate portions 302′, 304′, whichrespectively comprise first and second layers 302 a′, 302 b′ and 304 a′,304 b′. First and second layers 302 a′, 302 b′ define a channel 306′comprising an inlet 310′ and an outlet 312′. First and second layers 304a′, 304 b′ define a channel 308′ comprising an inlet 314′ and an outlet316′. First and second substrate portions 302′, 304′ were mated usingadhesive 324′ so that outlet 312′ communicated with inlet 314′ with afilter 318′ positioned therebetween. A portion of outlet 312′ was filedwith the activated beads prepared above to provide a processing region320′ comprising a retention member (the beads). A pipette 322′ (FIG. 14)secured by adhesive 326′ facilitated sample introduction.

In use, sample introduced via inlet 310′ passed along channel andthrough processing region 320′. Excess sample material passed alongchannel 308′ and exited device 300′ via outlet 316′. Polynucleotideswere preferentially retained by the beads as compared to inhibitors.Once sample had been introduced, additional liquids, e.g., a wash liquidand/or a liquid for use in releasing the retained polynucleotides wereintroduced via inlet 326′.

Retention of DNA

Retention of polynucleotides by the poly-L-lysine modified beads ofdevice 300′ was demonstrated by preparing respective devices comprisingprocessing regions having a volume of about 1 mL including about 1000beads. The beads were modified with poly-L-lysine of between about15,000 and 30,000 Da. Each processing region was filled with a liquidcomprising herring sperm DNA (about 20 uL of sample with a concentrationof about 20 mg/mL) thereby placing the beads and liquid in contact.After the liquid and beads had been in contact for 10 minutes, theliquid was removed from each processing region and subjected toquantitative real-time PCR to determine the amount of herring sperm DNApresent in the liquid.

Two controls were performed. First, an otherwise identical processingregion was packed with unmodified beads, i.e., beads that were identicalwith the poly-L-lysine beads except for the activation and poly-L-lysineincubation steps. The liquid comprising herring sperm DNA was contactedwith these beads, allowed to stand for 10 minutes, removed, andsubjected to quantitative real-time PCR. Second, the liquid comprisingthe herring sperm DNA (“the unprocessed liquid”) was subjected toquantitative real-time PCR.

Referring to FIG. 15, the first and second controls exhibitedessentially identical responses indicating the presence of herring spermDNA in the liquid contacted with the unmodified beads and in theunprocessed liquid. The liquid that had contacted the 3,000poly-L-lysine beads exhibited a lower response indicating that themodified beads had retained substantially all of the herring sperm DNA.The PCR response of the liquid that had contacted the 300,000 Dapoly-L-lysine beads exhibited an amplification response that was atleast about 50% greater than for the 3,000 Da beads indicating that thelower molecular weight surface modification was more efficient atretaining the herring sperm DNA.

Releasing DNA from Poly-L-Lysine Modified Beads

Devices having processing regions were packed with 3,000 Dapoly-L-lysine modified beads. Liquid comprising polynucleotides obtainedfrom group B streptococci (GBS) was contacted with the beads andincubated for 10 minutes as above for the herring sperm DNA. This liquidhad been obtained by subjecting about 10,000 GBS bacteria in 10 μl of 20mM Tris pH 8, 1 mM EDTA, 1% Triton X-100 buffer to thermal lysing at 97°C. for 3 min.

After 10 minutes, the liquid in contact with the beads was removed byflowing about 10 μl of wash solution (Tris-EDTA pH 8.0 with 1% Triton X100) through the processing region. Subsequently, about 1 μl of 5 mMNaOH solution was added to the processing region. This process left thepacked processing region filled with the NaOH solution in contact withthe beads. The solution in contact with the beads was heated to 95° C.After 5 minutes of heating at 95° C., the solution in contact with thebeads was removed by eluting the processing region with a volume ofsolution equal to three times the void volume of the processing region.

Referring to FIG. 16, five aliquots of solution were subjected toquantitative real-time PCR amplification. Aliquots E1, E2, and E3 eachcontained about 1 μl of liquid. Aliquot L was corresponds to liquid ofthe original sample that had passed through the processing region.Aliquot W was liquid obtained from wash solution without heating.Aliquot E1 corresponds to the dead volume of device 300, about equal tothe volume of channel 308. Thus, liquid of aliquot E1 was present inchannel 308 and not in contact with the beads during heating. Thisliquid had passed through the processing region prior to heating.Aliquot E2 comprises liquid that was present within the processingregion and in contact with the beads during heating. Aliquot E3comprises liquid used to remove aliquot E2 from the processing region.

As seen in FIG. 16, more than 65% of the GBS DNA present in the initialsample was retained by and released from the beads (Aliquot E2). AliquotE2 also demonstrates the release of more than 80% of the DNA that hadbeen retained by the beads. Less than about 18% of the GBS DNA passedthrough the processing region without being captured. The wash solutionwithout heating comprised less than 5% of the GBS DNA (Aliquot W).

Separation of Polynucleotides and Inhibitors

Buccal cells from the lining of the cheeks provide a source of humangenetic material (DNA) that may be used for single nucleotidepolymorphism (SNP) detection. A sample comprising buccal cells wassubjected to thermal lysing to release DNA from within the cells. Device300 was used to separate the DNA from concomitant inhibitors asdescribed above. A cleaned-up sample corresponding to aliquot E2 of FIG.16 was subjected to polymerase chain reaction. A control or crude sampleas obtained from the thermal lysing was also amplified.

Referring to FIG. 17, the cleaned-up sample exhibited substantiallyhigher PCR response in fewer cycles than did the control sample. Forexample, the clean-up sample exceeded a response of 20 within 32 cycleswhereas the control sample required about 45 cycles to achieve thesample response.

Blood acts as a sample matrix in variety of diagnostic tests includingdetection of infectious disease agents, cancer markers and other geneticmarkers. Hemoglobin present in blood samples is a documented potentinhibitor of PCR. Two 5 ml blood samples were lysed in 20 mM Tris pH 8,1 mM EDTA, 1% SDS buffer and introduced to respective devices 300, whichwere operated as described above to prepare two clean-up samples. Athird 5 ml blood sample was lysed and prepared using a commercial DNAextraction method Puregene, Gentra Systems, MN. The respectivecleaned-up samples and sample subjected to the commercial extractionmethod were used for a Allelic discrimination analysis (CYP2D6*4reagents, Applied Biosystems, CA). Each sample contained an amount ofDNA corresponding to about 1 ml of blood.

Referring to FIG. 18, the cleaned-up and commercially extracted samplesexhibited similar PCR response demonstrating that the processing regionof device 300′ efficiently removed inhibitors from the blood samples.

Protease Resistant Retention Member

The preparation of polynucleotide samples for further processing oftenincludes subjecting the samples to protease treatment in which aprotease cleaves peptide bonds of proteins in the sample. An exemplaryprotease is pronase, a mixture of endo- and exo-proteases. Pronasecleaves most peptide bonds. Certain ligands, such as poly-L-lysine aresusceptible to rupture by pronase and other proteases. Thus, if samplesare generally not subjected to protease treatment in the presence of theretention member if the ligands bound thereto are susceptible to theproteases.

Poly-D-lysine, the dextro enantiomer of poly-lysine resists cleavage bypronase and other proteases. The ability of a retention membercomprising bound poly-D-lysine to retain DNA even when subjected to aprotease treatment was studied.

Eight (8) samples were prepared. A first group of 4 samples contained1000 GBS cells in 10 μl buffer. A second group of 4 samples contained100 GBS cells in 10 μl buffer. Each of the 8 samples was heated to 97°C. for 3 min to lyse the GBS cells. Four (4) sample sets were createdfrom the heated samples. Each sample set contained 1 sample from each ofthe first and second groups. The samples of each sample sets weretreated as follows.

Referring to FIG. 19 a, the samples of sample set 1 were subjected topronase incubation to prepare respective protein cleaved samples, whichwere then heated to inactivate the proteases. The protein-cleaved,heated samples were contacted with respective retention members eachcomprising a set of poly-L-lysine modified beads. After 5 minutes, therespective sets of beads were washed with 5 microliters of a 5 mM NaOHsolution to separate inhibitors and products of protein cleavage fromthe bound DNA. The respective sets of beads were each contacted with asecond aliquot of NaOH solution and heated to 80 (eighty) ° C. for 2minutes to release the DNA. The solutions with released DNA wereneutralized with an equal volume of buffer. The neutralized solutionswere analyzed to determine the efficiency of DNA recovery. The resultswere averaged and shown in FIG. 19 b.

The samples of sample set 2 were subjected to pronase incubation toprepare respective protein cleaved samples, which were then heated toinactivate the proteases. The protein-cleaved, heated samples werecontacted with respective retention members each comprising a set ofpoly-D-lysine modified beads. After 5 minutes, the respective sets ofbeads were washed with 5 microliters of a 5 mM NaOH solution to separateinhibitors and products of protein cleavage from the bound DNA. Therespective sets of beads were each contacted with a second aliquot ofNaOH solution and heated to 80 (eighty) ° C. for 2 minutes to releasethe DNA. The solutions with released DNA were neutralized with an equalvolume of buffer. The neutralized solutions were analyzed to determinethe efficiency of DNA recovery. The results were averaged and shown inFIG. 19 b.

The samples of sample set 3 were subjected to pronase incubation toprepare respective protein cleaved samples. The proteases were notdeactivated either thermally or chemically. The protein-cleaved sampleswere contacted with respective retention members each comprising a setof poly-L-lysine modified beads. After 5 minutes, the respective sets ofbeads were washed with 5 microliters of a 5 mM NaOH solution to separateinhibitors and products of protein cleavage from the bound DNA. Therespective sets of beads were each contacted with a second aliquot ofNaOH solution and heated to 80 (eighty) ° C. for 2 minutes to releasethe DNA. The solutions with released polynucleotides were eachneutralized with an equal volume of buffer. The neutralized solutionswere analyzed to determine the efficiency of DNA recovery. The resultswere averaged and shown in FIG. 19 b.

The samples of sample set 4 were subjected to pronase incubation toprepare respective protein cleaved samples. The proteases were notdeactivated either thermally or chemically. The protein-cleaved sampleswere contacted with respective retention members each comprising a setof poly-D-lysine modified beads. After 5 minutes, the respective sets ofbeads were washed with 5 microliters of a 5 mM NaOH solution to separateinhibitors and products of protein cleavage from the bound DNA. Therespective sets of beads were each contacted with a second aliquot ofNaOH solution and heated to 80 (eighty) ° C. for 2 minutes to releasethe DNA. The solutions with released polynucleotides were eachneutralized with an equal volume of buffer. The neutralized solutionswere analyzed to determine the efficiency of DNA recovery. The resultswere averaged and shown in FIG. 19 b.

As seen in FIG. 19 b, an average of more than 80% of DNA from the GBScells was recovered using sample set 4 in which the samples werecontacted with poly-D-lysine modified beads and subjected to pronaseincubation in the presence of the beads without protease inactivation.The recovery efficiency for sample set 4 is more than twice as high asfor any of the other samples. Specifically, the recovery efficienciesfor sample sets 1, 2, 3, and 4, were 29%, 32%, 14%, and 81.5%,respectively. The efficiencies demonstrate that high recoveryefficiencies can be obtained for samples subjected to proteaseincubation in the presence of a retention member that retains DNA.

Other embodiments are within the claims.

What is claimed is:
 1. A microfluidic device, comprising: a processingregion having a vertical central axis therethrough, the processingregion comprised of an inlet, a retention member, a filter, and anoutlet; said retention member being configured to preferentially retainone or more polynucleotides in a sample as compared to polymerase chainreaction inhibitors in the sample, wherein said retention membercomprises a plurality of polynucleotide binding particles, and saidplurality of binding particles having surfaces that comprise apoly-cationic polyamide bound thereto, further wherein the retentionmember is configured to retain polynucleotides from a samplepreferentially to inhibitors in the sample as the sample passes throughthe retention member from the processing region inlet to the processingregion outlet; wherein the vertical central axis of the processingregion is perpendicular to a horizontal plane of the microfluidicdevice; wherein the processing region outlet is disposed along thevertical central axis above the processing region inlet; wherein thefilter is configured to prevent the plurality of binding particles frompassing downstream of the processing region; a device inlet incommunication with, and upstream of, the processing region; a deviceoutlet in communication with, and downstream of, the processing region;and a detection region in fluid communication with and downstream of theretention member.
 2. The microfluidic device of claim 1, wherein theplurality of particles have a volume less than 5 microliters.
 3. Themicrofluidic device of claim 1, wherein the sample has a volume from 0.5microliters to 3 milliliters.
 4. The microfluidic device of claim 1,wherein the poly-cationic polyamide comprises at least one ofpoly-DL-ornithine, PEI, poly-L-lysine and poly-D-lysine.
 5. Themicrofluidic device of claim 1, wherein the filter is disposed betweenthe retention member and the processing region outlet.
 6. Themicrofluidic device of claim 1, further comprising at least one channelhaving at least one sub-millimeter cross-sectional dimension.
 7. Themicrofluidic device of claim 1, further comprising a valve, wherein thevalve includes a mass of thermally responsive substance that isrelatively immobile at a first temperature and more mobile at a secondtemperature.
 8. The microfluidic device of claim 1, further comprising agate, wherein the gate includes a mass of thermally responsive substancewhich is relatively immobile at a first temperature and which, uponheating to a second temperature, changes state.
 9. The microfluidicdevice of claim 1, further comprising a fluid reservoir configured tohold a liquid.
 10. The microfluidic device of claim 9, wherein thereservoir comprises a liquid having a pH of at least about
 10. 11. Themicrofluidic device of claim 9, wherein the device is configured tocontact the retention member with the liquid.
 12. The microfluidicdevice of claim 1, further comprising an actuator, wherein the actuatorcomprises a chamber having a mass of thermally expansive materialtherein, and a gas.
 13. The microfluidic device of claim 1, furthercomprising a hydrophobic vent, wherein the vent comprises a layer ofporous hydrophobic material.
 14. The microfluidic device of claim 1,having three layers of polymeric material.
 15. The microfluidic deviceof claim 1, thermally associated with an array of heat sources.
 16. Themicrofluidic device of claim 15, wherein at least one of the heatsources is configured to heat an aqueous liquid in contact with theretention member to at least about 65° C.
 17. The microfluidic device ofclaim 16, wherein the liquid is held in a pouch, isolated from thereservoir by a generally impermeable membrane.
 18. The microfluidicdevice of claim 17, wherein the pouch additionally comprises a piercingmember configured to puncture the impermeable membrane.
 19. Themicrofluidic device of claim 1, further comprising one or more PCRreagents stored as one or more lyophilized particles.
 20. Themicrofluidic device of claim 19, wherein the lyophilized particles havean average volume of about 5 microliters or less.
 21. The microfluidicdevice of claim 1, further comprising one or more lysing reagents storedas one or more lyophilized particles.
 22. The microfluidic device ofclaim 21, wherein the lyophilized particles have an average volume ofabout 35 microliters or less.
 23. The microfluidic device of claim 1,additionally comprising a lysing chamber and a waste chamber.
 24. Themicrofluidic device of claim 1, wherein the particles are made of apolymeric material selected from the group consisting of: polystyrene,latex polymers, polyacrylamide, and polyethylene oxide.
 25. Themicrofluidic device of claim 24, wherein the polymeric material ismodified to include one or more carboxylic groups and/or one or moreamino groups covalently bound to the polymeric material, wherein thegroups provide an attachment point for one or more ligands of thepoly-cationic polyamide.
 26. The microfluidic device of claim 1, whereinthe particles have an average diameter of between about 4 microns andabout 20 microns.
 27. The microfluidic device of claim 1, wherein theparticles are present in a density of about 10⁸ particles permilliliter.
 28. The microfluidic device of claim 1, wherein at leastsome of the particles are magnetic.